Infectious diseases among shrimp have taken a devastating toll on aquaculture production. Among the most harmful pathogens are viruses, bacteria, and protozoans, with viruses posing the greatest threat to shrimp survival rates. Bacterial and fungal infections in shrimp can usually be controlled effectively by applying available chemical treatments to shrimp populations in hatchery ponds or tanks. However, there are currently no effective chemicals or antibiotics to treat viral diseases. Other strategies used in handling shrimp disease problems include immunostimulation, vaccination, quarantine, and environmental management. These strategies are generally targeted at three elements: pathogens, host, and environment. Boosting the shrimp's natural defense system against pathogens is a non-specific approach to combating disease which does not improve the shrimp's ability to cope with future outbreaks of the same disease since shrimp and other invertebrates lack a memory immune response based on antibody production. The lack of basic information about shrimp immunology is also another impediment to the development of efficient strategies for combating viral diseases via traditional methods.
Viral diseases are the most devastating problem facing shrimp aquaculture. The four major viruses, including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), and infectious hypodermal and hematopoietic necrosis virus (IHHNV), pose the greatest threat to penaeid shrimp farming worldwide. The IHHNV was first detected in Hawaii in 1981, causing up to 90% mortality in juvenile shrimp, Litopenaeus stylirostris (Lightner et al., “Infectious Hypodermal and Hematopoietic Necrosis, a Newly Recognized Virus Disease of Penaeid Shrimp,” J. Invert. PathoL 42: 62-70 (1983)). This virus has since been reported to infect most Litopenaeus species (which was previously known as the Penaeus species), including the Pacific white shrimp, L. vannamei and the blue shrimp, L. stylirostris, causing tremendous economic losses worldwide (Brock, “An Overview of Diseases of Cultured Crustaceans in the Asia Pacific Region,” in Fish Health Management in Asia-Pacific. Report on a Regional Study and Workshop on Fish Disease and Fish Health Management, ADB Agriculture Department Report Series No. 1. Network of Aquaculture Centres in Asia-Pacific. Bangkok, Thailand, pp. 347-395 (1991); Flegel, “Major Viral Diseases of the Black Tiger Prawn (Penaeus Monodon) In Thailand,” in NRIA International Workshop, New approaches to viral diseases of aquatic animals, Kyoto, Japan. Jan. 21-24, 1997, National Research Institute of Aquaculture, Nansei, Mie 516-01, Japan pp. 167-189 (1997)). TSV has infected United States farms rearing Litopenaeus vannamei since 1992 and has caused more than 2 billion dollars in damage to aquaculture farms (Brock, “An Overview of Taura Syndrome, an Important Disease of Farmed Penaeus Vannamei,” in C. L. Browndy and J. S. Hopkins, (eds.), Swimming Through Troubled Water. Proceedings of the Special Section on Shrimp Farming, Baton-Rouge, La.: World Aquaculture Society pp. 84-94 (1995); Lightner et al., “Risk of Spread of Penaeid Shrimp Viruses in the Americas by the International Movement of Live and Frozen Shrimp,” Rev. Sci. Tech. 16(1):146-60 (1997)). In Hawaii, both TSV and IHHNV infections in shrimp farms have been frequently reported since 1994 (MacMillan, “Shrimp Diseases in Hawaii, USA”. UNIHI-SG-FS-96-02. University of Hawaii Sea Grant College Program, Honolulu (1996)). Controlling viral diseases clearly represents a great challenge as there are currently no effective chemicals or antibiotics to treat viral infection. The serious effects of viral disease outbreaks among cultured shrimp coupled with a decline in natural fisheries of healthy shrimp (Pullin et al., “Domestication of Crustaceans,” Asian Fisheries Sci. 11(1):59-69 (1998)), have led to a critical demand for advanced biotechnological applications.
The two major penaeid shrimp species cultured in the Americas, L. vannamei and L. Stylirostris, have differing susceptibilities to TSV and IHHNV. L. vannamei is more resistant to IHHNV, but susceptible to TSV, whereas L. stylirostris is innately resistant to TSV but highly susceptible to IHHNV (Lightner et al., “Strategies for the Control of Viral Diseases of Shrimp in the Americas,” Fish Pathology 33:165-180 (1998)). Despite the relative resistance of L. vannamei to IHHNV, runt deformity syndrome (RDS) was still observable in this shrimp species when exposed to IHHNV. Although these viral diseases may not be completely fatal, the reduced growth rate resulting from viral-induced RDS results in immense revenue losses for shrimp farmers each year.
Systematic genetic selection is known to enhance disease resistance in a number of farmed plants and animals, including fish (Gjedrem et al., “Genetic Variation in Susceptibility of Atlantic Salmon to Furunculosis,” Aquaculture 97:1-6 (1991)). However, the efficacy of breeding for disease resistance in penaeid shrimp is not well established because of the paucity of information about relevant genetic parameters, such as phenotypic and genetic variation, heritability, and genetic correlations between traits. In response to viral-disease problems facing the shrimp farming industry, the U.S. Marine Shrimp Farming Program (USMSFP), with funding from USDA/CSREES, has developed a selective breeding program to enhance disease resistance and improve growth in L. vannamei (Moss et al., “Breeding for Disease Resistance in Penaeid Shrimp: Experiences From the U.S. Marine Shrimp Farming Program,” In: Proceedings of the 1st Latin American Shrimp Farming Congress (D. E. Jory, ed.), Panama City, Panama, 9 pp. (1998); Argue et al., “Selective Breeding of Pacific White Shrimp (Litopenaeus Vannamei) for Growth and Resistance to Taura Syndrome Virus,” Aquaculture 204:447-460 (2002)). Although high between-family variation in response to TSV challenge was observed in all groups of shrimp tested, heritability estimates (h2) for TSV resistance were low (h2full-sib=0.14). Heritability describes the percentage of phenotypic variance that is inherited in a predictable manner and is used to determine the potential response to selection (Tave, “Genetics for Fish Hatchery Managers,” 2nd ed., AVI, New York, 415 pp (1993)). Estimates of h2 typically are low for fitness traits, such as disease resistance, and phenotypes with h2≦0.15 are difficult to improve by selection. Although the development of TSV-resistant strains of L. vannamei have benefited shrimp farmers, breeding for TSV resistance is not a panacea to the health problems plaguing the industry. Viruses can mutate, thereby rendering selectively bred shrimp incapable of defending themselves against new strains of virus. Furthermore, TSV resistance could be negatively correlated with resistance to other pathogens. There is also the potential to produce shrimp that respond well in disease-challenge tests used in breeding programs, but perform poorly when stocked in commercial ponds.
The use of molecular biology techniques to produce pathogen-resistant strains of shrimp through genetic transformation technology is considered a highly promising strategy for control of shrimp viral disease (Mialhe et al., “Future of Biotechnology-Based Control of Disease in Marine Invertebrates,” Mol. Mar. Biol. and Biotechnol. 4(4):275-83 (1995); Bachere et al., “Transgenic Crustaceans,” World Aquaculture 28(4):51-5 (1997)). In the past decade, pathogen-resistant transgenic animals and plants have been developed (Beachy, “Virus Cross-Protection in Transgenic Plants,” in D. P. S. Verma, and R. B. Goldberg, (eds.), Plant Gene Research: Temporal and Spatial Regulation of Plant Genes, New York: Springer Verlag pp. 313-327 (1998); Kim et al., “Disease Resistance in Tobacco and Tomato Plants Transformed with the Tomato Spotted Wilt Virus Nucleocapsid Gene,” Plant Dis. 78:615-21 (1993); Sin, “Transgenic Fish,” Rev. Fish Biol. 7(4):417-41 (1997)), but use of such technology has only just begun for shrimp research. While methods for detecting viral disease in shrimp, including polymerase chain reaction (Dhar et al., “Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Virus (WSV) of Shrimp by Real-Time Quantitative PCR and SYBR Chemistry,” J. Clin. Microbiol. 39:2835-2845 (2001); Tang et al., “Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus in Penaeid Shrimp by Real-Time PCR,” Dis. Aquat. Org. 44(2):79-85 (2001)), light microscopy, and transmission electron microscopy (Nunan et al., “Reverse Transcription Polymerase Chain Reaction (RT-PCR) Used for the Detection of Taura Syndrome Virus (TSV) in Experimentally Infected Shrimp,” Dis. Aquatic. Org. 34:87-91 (1998); Goarant et al., “Arbitrarily Primed PCR to Type Vibrio Spp. Pathogenic for Shrimp,” Appl. Environ. Microbiol. 65(3):1145-1151 (1999); Chen et al., “Establishment of Cell Culture Systems from Penaeid Shrimp and Their Susceptibility to White Spot Disease and Yellow Head Viruses,” Meth, in Cell Sci. 21:199-206 (1999); Toullec, “Crustacean Primary Cell Culture: a Technical Approach,” Meth. In Cell Sci. 21:193-8 (1999); Sukhumsirichart et al., “Characterization and PCR Detection of Hepatopancreatic Parvovirus (HPV) from Penaeus Monodon in Thailand,” Dis. Aquat. Org. 38:1-10 (1999) are widely used, these methods for controlling viral disease in shrimp are still in development. The first studies on genetic transformation of marine molluscs and shrimp were initiated in 1988 in France at IFREMER, in the United States at the University of Maryland Biotechnology Institute, and in Australia at CSIRO. A few studies on the introduction of foreign DNA into shrimp embryos via transfection methods have obtained preliminary data demonstrating transient expression of a reporter gene by heterologous promoters (Gendreau et al., “Transient Expression of a Luciferase Reporter Gene After Ballistic Introduction Into Artemia franciscana (Crustacea) Embryos,” Aquaculture 133:199-205 (1995)). Recent advances in gene transfer technology such as these hold immense potential for developing transgenic shrimp harboring genes that convey viral disease resistance or enhance shrimp growth rates. Gene transfer technology thus represents a practical alternative to the lengthy and expensive selective breeding process (Wolfus et al., “Application of the Microsatellite Technique for Analyzing Genetic Diversity in Shrimp Breeding Programs,” Aquaculture 152:35-47 (1997)) and provides a powerful tool for revolutionizing not only shrimp aquaculture, but also livestock husbandry in general.
Construction of an effective expression vector is an important step toward implementing the genetic transformation process in animals. The expression vector is generally composed of three elements: a promoter, a target gene, and a region having transcriptional termination signals. Among these three components, a suitable promoter is an essential element for a successful gene transformation system. The promoter determines where, when, and under what conditions the target gene should be turned on.
A suitable promoter that is appropriate for aquaculture and acceptable to consumers should ideally be derived from marine origin and should not pose any potential health hazards. Several fish gene promoters have been successfully isolated and used to drive foreign gene expression in fish (Jankowski et al., “The GC Box as a Silencer,” Biosci. Rep. 7:955-63 (1987); Zafarullah et al., “Structure of the Rainbow Trout Metallothionein B Gene and Characterization of its Metal-Responsive Region,” Mol. Cell. Biol. 8:4469-76 (1988); Liu et al., “Development of Expression Vectors for Transgenic Fish,” Bio/Technology 8:1268-1272 (1990b); Gong et al., “Functional Analysis and Temporal Expression of Promoter Regions From Fish Antifreeze Protein Genes in Transgenic Japanese Medaka Embryos,” Mol. Mar. Biol. Biotechnol. 1(1):64-72 (1991); Du et al., “Growth Enhancement in Transgenic Atlantic Salmon by the Use of Fish Antifreeze/Growth Hormone Chimeric Gene Constructs,” Biotechnology 10:176-81 (1992); Gong et al., “Transgenic Fish in Aquaculture and Developmental Biology,” Current Topic in Develop. Biol. 30:175-213 (1995); Chen et al., “Transgenic Fish and Aquaculture,” Biotechnol. Apl. 13(1):50 (1996); Chan et al., “PCR Cloning and Expression of the Molt-Inhibiting Hormone Gene for the Crab (Charybdis feriatus),” Gene 224:23-33 (1998); Gong, “Zebrafish Expressed Sequence Tags and Their Applications,” Meth. Cell Biol. (zebrafish volume) 60:213-233 (1998); Ju et al., “Faithful Expression of Green Fluorescent Protein (GFP) in Transgenic Zebrafish Embryos Under Control of Zebrafish Gene Promoters,” Dev. Genet. 25(2):158-67 (1999); Yoshizaki et al., “Germ Cell-Specific Expression of Green Fluorescent Protein in Transgenic Rainbow Trout Under Control of the Rainbow Trout Vasa-Like Gene Promoter,” Int. J. Dev. Biol. 44(3):323-6 (2000)). Other promoters used to date in transgenic marine fish include mouse metallothionein (McEvoy et al., “The Expression of a Foreign Gene in Salmon Embryos,” Aquaculture 68:27-37 (1988); Rahman et al., “Fish Transgene Expression by Direct Injection Into Fish Muscle,” Mol. Mar. Biol. Biotechnol. 1:286-289 (1992)), heat shock promoters (Bayer et al., “A Transgene Containing lacZ is Expressed in Primary Sensory Neurons in Zebrafish,” Development 115:421-446 (1992); Krone, “Several Unique Hsp 90 Genes are Expressed During Embryonic Development of Zebrafish,” Presented at Symposium on Advances in Molecular Endocrinology of Fish, May 23-25, Toronto, Canada (1993)), chicken β-actin promoter (Lu et al., “Integration and Germline Transmission of Human Growth Hormone Gene in Medaka (Oryzias latipes),” presented at Second International Marine Biotechnology Conference, 1991, Baltimore, Md. (1991); Inoue et al., “Introduction, Expression, and Growth-Enhancing Effect of Rainbow Trout Growth Hormone cDNA Fused to an Avian Chimeric Promoter in Rainbow Fry,” J. Mar. Biotechnol. 1:131-4 (1993)), carp β-actin promoter (Liu et al., “Functional Analysis of Elements Affecting Expression of the β-Actin Gene of Carp,” Mol. Cell Biol. 10:3432-3440 (1990); Rahman et al., “Fish Transgene Expression by Direct Injection Into Fish Muscle,” Mol. Mar. Biol. Biotechnol. 1:286-289 (1992)), the antifreeze protein promoter from the ocean pout (Macrozoarces americanus) (Gong et al., “Functional Analysis and Temporal Expression of Promoter Regions From Fish Antifreeze Protein Genes in Transgenic Japanese Medaka Embryos,” Mol. Mar. Biol. Biotechnol. 1(1):64-72 (1991); Hew et al., “Antifreeze Protein Gene Transfer in Atlantic Salmon,” Presented at Second International Marine Biotechnology Conference, 1991, Baltimore, Md. (1991); Du et al., “Growth Enhancement in Transgenic Atlantic Salmon by the Use of Fish Antifreeze/Growth Hormone Chimeric Gene Constructs,” Biotechnology 10:176-81 (1992)), and the histone promoter from the trout (Muller et al., “Introducing Foreign Genes Into Fish Eggs With Electroporated Sperm as a Carrier,” Mol. Mar. Biol. Biotechnol. 1:276-281 (1992)). Unfortunately, these promoters have disadvantages, including inconsistent transgenic expression, potential toxicity due to their viral origin, and association with metabolic poisons and/or tumor-inducing sequences, all of which will present major stumbling blocks toward attaining FDA approval for the commercial use of transgenic animals. However, isolation and use of promoter genes from crustacean shrimp has not been reported. Thus, the tremendous potential presented by gene transfer technology has not yet been realized in shrimp aquaculture due to the lack of a constitutive, non-inducible, and non-developmentally regulated promoter to efficiently drive the expression of heterologous genes in shrimp and other marine animals.
The present invention is directed to overcoming these and other deficiencies in the art.